Molecular analysis of genetic recombination and DNA break repair

 描述(申请人提供)：本研究的长期目标是确定同源基因重组和DNA断裂修复的分子机制。这一目标是通过结合突变的遗传分析和细胞中蛋白质和DNA的生化分析来实现的。本研究利用裂殖酵母、裂殖酵母、大肠杆菌及其噬菌体Lambda。所有这些都是经过广泛研究的高度易驯化的模式生物，具有包括人类在内的所有生物的共同特征。这些研究集中在庞氏链霉菌的减数分裂重组，其高重组率有利于遗传和生化分析，以及细菌中重组和DNA断裂修复的主要途径(RecBCD)。在以往成果的基础上，这项研究目前集中在以下目标上。1)确定减数分裂重组如何在着丝粒和着丝粒附近被重新抑制，以防止染色体错误分离、不孕和出生缺陷。2)确定如何为减数分裂DNA断裂修复选择正确的模板(姊妹或同源)，以阐明如何在整个基因组中控制交换以有利于适当的减数分裂染色体分离。3)确定合适的联合DNA分子是如何形成的，并避免不适当的DNA分子，以确保减数分裂产物(配子)的高活性。4)确定RecBCD酶与其在DNA上的控制部位(CHI热点)相互作用的机制，以便进行高清晰度的细菌重组分析。5)确定最近发现的小分子抑制剂如何阻止一种或另一种RecBCD酶活性，以开发成潜在有用的、迫切需要的抗生素。这些目标将通过对突变的遗传分析、细胞内蛋白质和染色体位置的荧光显微镜、减数分裂细胞DNA中间体的物理分析以及分离蛋白质的酶和生物物理分析的组合来实现。这些研究的结果将阐明重组和DNA断裂修复的分子机制，以及确保重组发生在染色体上适当的时间和地点的重组控制。重组对于有效的减数分裂染色体分离，对于经常出现的DNA双链断裂的无错误修复，以及对产生细胞和生物多样性是重要的。重组的缺失可产生染色体重排，如易位、重复和缺失，这些通常与不孕不育、出生缺陷和癌症有关或导致不孕、出生缺陷和癌症。细菌DNA断裂修复的抑制剂是一种很有前途的新型抗生素，是对抗日益流行的耐药细菌的迫切需要。

 DESCRIPTION (provided by applicant): The long-term goal of the research proposed here is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This objective is approached by a combination of genetic analysis of mutants and biochemical analysis of proteins and DNA from cells. This research uses the fission yeast Schizosaccharomyces pombe as well as the bacterium Escherichia coli and its phage lambda. All are widely studied, highly tractable model organisms with features common to all organisms, including humans. The studies are focused on meiotic recombination in S. pombe, whose high rates of recombination facilitate both genetic and biochemical analyses, and on the major (RecBCD) pathway of recombination and DNA break repair in bacteria. Building on past achievements, the research is currently focused on the following goals. 1) Determine how meiotic recombination is repressed in and near centromeres, to prevent chromosome missegregation, infertility, and birth defects. 2) Determine how the correct template (sister or homolog) is chosen for meiotic DNA break repair, to elucidate how crossing-over is controlled across the genome to favor proper meiotic chromosome segregation. 3) Determine how appropriate joint DNA molecules are formed, and inappropriate ones are avoided, to ensure high viability of meiotic products (gametes). 4) Determine the mechanism by which RecBCD enzyme interacts with its controlling site on DNA (the Chi hotspot), to allow high-definition analysis of bacterial recombination. 5) Determine how recently discovered small-molecule inhibitors block one or another RecBCD enzymatic activity, to be developed into potentially useful, sorely needed antibiotics. These goals will be attacked by a combination of genetic analysis of mutants, fluorescence microscopy of intracellular proteins and chromosomal sites, physical analysis of DNA intermediates from meiotic cells, and enzymatic and biophysical analyses of isolated proteins. The results of these studies will elucidate the molecular mechanism of recombination and DNA break repair as well as the controls on recombination that ensure that it occurs at the proper time and place along chromosomes. Recombination is important for faithful meiotic chromosome segregation, for error-free repair of frequently arising DNA double-strand breaks, and for generating cellular and organismal diversity. Aberrancies of recombination can generate chromosomal rearrangements, such as translocations, duplications, and deletions, which are often associated with or the cause of infertility, birth defects, and cancers. Inhibitors of bacterial DNA break repair are promising novel antibiotics, which are desperately needed to combat the ever-increasing prevalence of drug-resistant bacteria.